Anti-IgM antibody-induced cell death in a human B lymphoma cell line, B104, represents a novel programmed cell death.

We investigated the mechanisms of anti-IgM antibody-induced cell death in a recently established human surface IgM+ IgD+ B lymphoma cell line, B104, the growth of which is irreversibly inhibited by anti-IgM antibody but not by anti-IgD antibody, and compared it with the cell death of T cells via TCR/CD3 complex and with the cell death of a murine anti-IgM antibody-sensitive B lymphoma cell line, WEHI-231. The rapid time course of B104 cell death and its requirements for de novo macromolecular synthesis and Ca2+ influx suggest that anti-IgM antibody-induced B104 cell death is an active Ca(2+)-dependent programmed cell death. Moreover, cyclosporin A rescued B104 cells from this lethal signal, via surface IgM, suggesting that the intracellular mechanisms involved are quite similar to those of T cell death. DNA fragmentation, which has been reported in TCR/CD3 complex-mediated T cell death, apoptosis, was not involved in the B104 cell death process, but the possible involvement of DNA single-strand breaks was suggested. Observations under light microscopy and transmission electron microscopy indicated that the morphologic features of dying B104 cells resembled necrosis rather than apoptosis. B104 cell death was shown to be quite distinct from that of WEHI-231 in cell death kinetics, the mode of cell death, and the response to cyclosporin A. These data collectively indicate that the death of B104 cells resulting from surface IgM cross-linking represents a hitherto undefined mode of programmed cell death.     

Anti-IgM but not anti-IgD antibodies inhibit cell division of normal human mature B cells.

Insolubilized anti-IgD antibody markedly increased DNA synthesis in and cell division of normal peripheral blood B cells (PBL-B) when used in combination with IL-4. Anti-IgM antibodies also induced DNA synthesis of PBL-B, but their ability to induce cell division was less than that of anti-IgD antibodies even when used in combination with IL-4. Moreover, anti-IgM antibodies inhibited cell division of PBL-B stimulated with insolubilized anti-IgD antibody plus IL-4 without affecting DNA synthesis. Anti-IgM antibodies also inhibited Staphylococcus aureus Cowan I-induced cell division of PBL-B without affecting DNA synthesis. These results indicate that cross-linkage of surface IgM (sIgM) in mature B cells generates negative signals to inhibit cell division of mature B cells. Because anti-IgD antibodies did not inhibit cell division at all, the role of sIgD in the regulation of cell division of mature B cells may be quite different from that of sIgM. IFN-alpha/beta promoted cell division of PBL-B stimulated with insolubilized anti-IgD antibody plus IL-4. They also counteracted the inhibitory effect of anti-IgM antibody on cell division of PBL-B.     

Development of formulae for estimating amylose content and resistant starch content based on the pasting properties measured by RVA of Japonica polished rice and starch

We searched for the easy and simple method to measure the novel indicators which reflect not only AAC, but also (RS) based on pasting properties using RVA. Novel indexes such as SB/Con and Max/Fin (Maximum viscosity/Minimum viscosity) ratios had a very high correlation with proportion of intermediate and long chains of amylopectin; Fb₁₊₂₊₃ (DP ≧ 13). In Japonica polished rice, estimation formulae for AAC and RS content were developed using novel indexes based on pasting properties by RVA, and these equations showed determination coefficients of 0.89 and 0.80 for calibration and 0.71 and 0.75 for validation test. We developed the estimation formulae for AAC and RS content for Japonica starch samples. These equations showed determination coefficients of 0.86 and 1.00 for calibration and 0.76 and 0.83 for validation test, which showed that these equations can be applied to the unknown rice samples.     

GABA-gated chloride ion influx in brains of epileptic El mice

GABA-gated chloride ion influx was measured in brain microsac preparations of epileptic El mice. There was significantly greater sensitivity to GABA in stimulated El mice (which had 14–18 convulsions induced at weekly intervals) than in unstimulated El mice (which had not experienced convulsions) or ddY mice. GABA-gated chloride ion influx was significantly decreased 20 min after a single convulsion, and returned to the preconvulsion level 60 min after a convulsion. These findings suggest that the functional state of GABA-gated chloride channel in El mice is changed secondarily by single or repeated convulsions.     

Identification of HeLa cell glucose 6-phosphate dehydrogenase

Although the electrophoretic mobility of HeLa G6PD is similar to that of the common Negro variant G6PD A+, several reports have suggested slight differences between HeLa G6PD and G6PD A+. This study, carried out using the pure homogeneous B+, A+, and HeLa G6PD, showed that (1) the electrophoretic mobility of HeLa G6PD is identical to that of G6PD A+, (2) the enzymatic properties and thermostability of HeLa G6PD are indistinguishable from those of G6PD A+, and (3) the peptide map of the tryptic digest of HeLa G6PD is identical to that of G6PD A+, with one peptide spot of HeLa G6PD different from the corresponding spot of G6PD B+. These results indicate that the structure of HeLa G6PD is identical to that of G6PD A+, and that the amino acid substitution in HeLa G6PD is from one asparagine residue in the wild-type G6PD B+ to an aspartic acid residue in HeLa G6PD.This research was supported by research grant GM 15253 from the National Institutes of Health.     

A quantitative enzyme-linked immunosorbent assay for Dermatophagoides pteronyssinus-specific immunoglobulin G

A monoclonal mouse anti-human IgG was used to develop an enzyme-linked immunosorbent assay (ELISA) for the measurement of Dermatophagoides pteronyssinus (DP)-specific IgG in human sera. This monoclonal antibody (HG2-25) binds to all subclasses of IgG but not to IgA, IgM, or IgE. For the assay, the DP antigen is coated firmly on polystyrene beads through physical adsorption and any leakable antigen is washed off. The assay gives satisfactory reproducibility and parallelism of the dilution curves. Using 0.1% human serum albumin as a substitute for the DP-specific IgG preabsorbed diluent gave extremely low backgrounds and high sensitivity. Horseradish peroxidase-labeled HG2-25 prepared with the optimum degree of conjugation and free of polymerized conjugates gave responses fairly proportional to the doses. This ELISA gives a satisfactory recovery and is not affected by nonspecific IgG levels in human sera.     

Isolation and Identification of Nicotinic Acid as a Flower-Inducing Factor in Lemna

Extracts of flowering plants of the long-day plant Lemna gibbaG3 and the short-day plants Lemna paucicostata 151 and 381 weretested on L. paucicostata 151 for flower-inducing activity.Crude extracts failed to show any activity but after severalpurification steps three fractions with flower-inducing activitywere obtained. One fraction obtained from all three plants wasshown to contain nicotinic acid by mass spectroscopic and NMRspectroscopic analyses. These results raise the possibilitythat nicotinic acid may act to influence the flowering processin Lemna. (Received August 28, 1985; Accepted October 29, 1985)     

Regulation of the expression of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) on a human eosinophilic leukemia cell line EoL-3.

The effects of several cytokines and phorbol myristate acetate (PMA) on LFA-1 and ICAM-1 expression on a human eosinophilic leukemia cell line, EoL-3, were investigated and compared with those of a human monocytic leukemia cell line, U937. EoL-3 cells expressed large amounts of LFA-1 and small amounts of ICAM-1, and their expression was regulated similarly in EoL-3 cells and U937 cells. Interferon-gamma (IFN-gamma) enhanced ICAM-1 expression but not LFA-1 expression, and PMA augmented both LFA-1 and ICAM-1 expression. IFN-gamma and PMA showed an additive effect on ICAM-1 expression. These results collectively suggest that expression of LFA-1 and ICAM-1 is regulated differently and that IFN-gamma and PMA regulate the expression through different mechanisms. PMA but not IFN-gamma induced homotypic adhesion of EoL-3 and U937 cells, suggesting that PMA but not IFN-gamma activated the adhesive function of these cells. Staurosporin, an inhibitor of protein kinases (PKs), partly suppressed IFN-gamma- and PMA-augmented expression of ICAM-1 on EoL-3 and U937 cells, but did not affect PMA-augmented LFA-1 expression, suggesting that staurosporin-sensitive PKs are involved in IFN-gamma- and PMA-augmented ICAM-1 expression but not in PMA-augmented LFA-1 expression. The role of protein kinase C (PK-C) in these mechanisms was not revealed because a PK-C inhibitor, H-7, did not show any definitive effect on IFN-gamma- and PMA-induced expression of LFA-1 and ICAM-1. Moreover, cyclic AMP (cAMP)- and cGMP-dependent pathways were not shown to be involved in the augmentation of the expression of these molecules.